Skeletal muscle is a site of insulin resistance after denervation and in pathological states characterized by hyperinsulinemia, hyperglycemia and/or persistent elevations of plasma free fatty acid (FFA). This proposal will examine the hypothesis that insulin resistance in these situations involves alterations in a diacylglycerol-protein kinase C (DAG-PKC) signalling system. Based on preliminary data with an incubated soleus muscle preparation, we are proposing a model in which increases in DAG in these conditions (1) occur in a specific pool, (2) are predominantly due to DAG synthesis de novo and (3) are associated with an increase in PKC, and ultimately with alterations In DAG-PKC signalling that result in insulin resistance. The proposed studies will both test this paradigm and explore the biological role of the DAG-PKC signalling system in insulin action. Using incubated and perfused muscle preparations, we will carry out studies with the following aims: 1. To determine the mechanisms for the increase in DAG synthesis in insulin+glucose-stimulated and denervated soleus muscles. 2. To characterize the interrelationships between changes in DAG mass and synthesis and PKC activity. 3. To characterize the temporal relations between changes in DAG-PKC signalling and the development of insulin resistance. 4. To determine whether alterations in DAG-PKC affect insulin-mediated gene expression. 5. To compare DAG-PKC signalling in the soleus , a predominantly slow-twitch red muscle, and the extensor digitorum longus (EDL), a muscle composed mainly of white fibers. 6. To evaluate the effect of prior exercise, a maneuver that increases insulin sensitivity in muscle, on DAG-PKC signalling. These studies should provide novel information about the linkage between fuel-metabolism and signal transduction in skeletal muscle. They should also yield new insights into the role of DAG-PKC signalling in the pathogenesis of insulin resistance.